C4B gene inhibitor composition and application thereof

Abstract

The invention provides a C4B gene inhibitor composition and application thereof, and belongs to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a positive-sense strand and an antisense strand, the positive-sense strand comprises at least 15 continuous nucleotides in any sequence as shown in SEQ ID NO: 1-SEQ ID NO: 180 or a nucleotide sequence, and the difference between the at least 15 continuous nucleotides and the nucleotide sequence is not more than 3 nucleotides; and / or the antisense strand comprises at least 15 consecutive nucleotides in any sequence as shown in SEQ ID NO: 181-SEQ ID NO: 360 or a nucleotide sequence which is different from the at least 15 consecutive nucleotides by not more than 3 nucleotides. The double-stranded oligonucleotide can inhibit the expression of the complement C4B gene of mammals, and achieves the effects of relieving, treating and / or preventing complement C4B-mediated diseases or symptoms.

Description

C4B gene inhibitor composition and application thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310970501.1 filed with the State Intellectual Property Office of China on August 3, 2023, and entitled “C4B gene inhibitor composition and its application,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the technical field of nucleic acid drugs, and in particular relates to an RNAi composition for inhibiting the expression of complement C4B gene, such as a double-stranded ribonucleic acid (dsRNA) composition and its application. Background Art

[0004] The complement system is part of the host's innate immune system and is composed of multiple proteins, present in the blood as soluble or membrane-associated proteins. Complement C4 is a multifunctional β1-globulin. During activation of the traditional complement pathway, C4 is hydrolyzed by C1s into C4a and C4b, which play a role in complement activation, promoting phagocytosis, preventing immune complex deposition, and neutralizing viruses. Inappropriate activation of the complement system contributes to the proliferation and / or initiation of lesions in many diseases. Inhibition of excessive or dysregulated complement activation can treat or alleviate related diseases or conditions.

[0005] Therefore, there is a need in the art to provide a complement C4B inhibitor composition for treating diseases or disorders related to complement gene C4B by regulating its activity.

[0006] Summary of the Invention

[0007] The present disclosure provides a complement C4B inhibitor, comprising a double-stranded oligonucleotide, a double-stranded RNAi agent, and a pharmaceutical composition comprising the same; the present disclosure provides a method for treating, preventing, or alleviating a disease associated with a disorder of the alternative complement pathway in a subject by administering a complement C4B inhibitor. The present disclosure also provides a method for inhibiting C4B expression by administering a C4B-specific inhibitor (double-stranded RNAi agent) to a subject.

[0008] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0009] In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for inhibiting complement C4B gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or the antisense strand comprising at least 15 consecutive nucleotides of any one of SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides.

[0010] Each nucleotide of the double-stranded oligonucleotide is an unmodified or modified nucleotide.

[0011] In some optional embodiments of the present disclosure, at least one nucleotide in the double-stranded oligonucleotide is modified.

[0012] In some optional embodiments of the present disclosure, all nucleotides in the sense strand and all nucleotides in the antisense strand of the double-stranded oligonucleotide are modified nucleotides.

[0013] The modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more nucleotides substituted with OR; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0014] In a second aspect of the present disclosure, the present disclosure provides a double-stranded RNAi agent for inhibiting complement C4B gene expression, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, and the double-stranded region is represented by the following formula (I):

[0015] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0016] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I),

[0017] Among them, SS represents the sense strand and AS represents the antisense strand;

[0018] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0019] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy-modified nucleotide;

[0020] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0021] Said a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; and c is selected from an integer of 6-10.

[0022] In a specific embodiment of the present disclosure, the sense strand is any modified sense strand nucleotide sequence shown in Table 2-3, and / or the antisense strand is any modified antisense strand nucleotide sequence shown in Table 2-3.

[0023] In some embodiments, the double-stranded RNAi agent further comprises a ligand; in some optional embodiments of the present disclosure, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent.

[0024] In some embodiments, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives connected by a linker selected from a monovalent, divalent or trivalent branched linker.

[0025] In some embodiments, the ligand structure is as follows:

[0026] * represents the attachment site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is independently selected from hydroxyl or sulfhydryl; p is independently selected from 1, 2 or 3; q is independently selected from 1, 2 or 3; each R3 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is C1-C 20The alkylene chain, or one or more C atoms in the chain, is substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is independently NH, O or S.

[0027] In other embodiments, the specific structure of the ligand is as follows:

[0028] In some embodiments, the 3' end of the sense strand of the double-stranded RNAi agent is covalently coupled to a ligand.

[0029] In a third aspect of the present disclosure, the present disclosure provides use of any of the following in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the complement C4B gene:

[0030] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0031] (2) The double-stranded RNAi agent described in the second aspect of the present disclosure.

[0032] In a fourth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any one of the following, and optionally a pharmaceutically acceptable carrier or excipient:

[0033] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0034] (2) The double-stranded RNAi agent described in the second aspect of the present disclosure.

[0035] In a fifth aspect of the present disclosure, the present disclosure provides a kit comprising any one of the following:

[0036] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0037] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0038] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0039] In a sixth aspect of the present disclosure, the present disclosure provides a method for inhibiting complement C4B expression in cells in vitro, the method comprising:

[0040] (a) contacting the cell with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition thereof; and

[0041] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the complement C4B gene, thereby inhibiting expression of the complement C4B gene in the cells.

[0042] In a seventh aspect of the present disclosure, the present disclosure provides a method for alleviating, treating and / or preventing a disease or condition mediated by complement C4B, characterized by administering any of the following to a subject:

[0043] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0044] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0045] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0046] Among them, the complement C4B-mediated diseases or conditions include diseases related to the mRNA level of complement C4B gene expression, including IgA nephropathy, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), glomerulopathy, systemic lupus erythematosus, etc.

[0047] In an eighth aspect of the present disclosure, the present disclosure provides a method for inhibiting complement C4B gene expression, characterized in that the method comprises administering any of the following to a subject:

[0048] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0049] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0050] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0051] In some alternative embodiments of the present disclosure, the subject is a human or other mammal.

[0052] The double-stranded RNAi agent or pharmaceutical composition thereof provided by the present disclosure can effectively regulate the expression level of the complement C4B gene, and thus can effectively treat and / or prevent diseases or symptoms associated with dysregulated complement C4B gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 shows the inhibitory activity of target genes in primary mouse liver cells after administration of double-stranded RNAi agents in Example 2.

[0054] FIG2 shows the inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi agents in Example 3.

[0055] FIG3 shows the inhibitory activity of target genes in HepG2 cells after administration of double-stranded RNAi agents in Example 4. DETAILED DESCRIPTION

[0056] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Those skilled in the art can refer to the contents of this document and appropriately improve the process parameters for implementation.

[0057] Explanation of terms

[0058] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as those generally understood by those of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. Publications, patent applications, patents and other references referred to herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting.

[0059] In this disclosure, the terms "include," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns with an indefinite number also include plural referents.

[0060] In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0061] The term "linked," when referring to a connection between two molecules, means that the two molecules are joined by a covalent bond or that the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond).

[0062] In the present disclosure, "fluorinated nucleotides" or "2'-fluorinated modified nucleotides" refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-fluorinated modified nucleotides" refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. The "methoxy-modified nucleotides" or "2'-O-methoxyethyl-modified nucleotides" refer to nucleotides in which the 2' hydroxyl group of the ribose group is replaced by a methoxy group or a methoxyethyl group. "Methoxy-modified nucleotides" are also recorded as 2'-OMe or 2'-O-methyl modified nucleotides, which can be used interchangeably. "2'-O-methoxyethyl-modified nucleotides" are also recorded as 2'-MOE modifications. 2'-deoxynucleotides refer to nucleotides in which the 2' position of the ribose group is hydrogen.

[0063] In the present disclosure, the term "halogen" or "halo" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0064] In this disclosure, the term "C1-C6 alkyl" refers to straight-chain and branched alkyl groups containing from 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbon atoms is named, it is intended to encompass all branched and straight-chain forms having that number of carbon atoms. For example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl.

[0065] In the present disclosure, the term "C1-C6 alkoxy" refers to straight-chain and branched alkoxy groups containing 1 to 6 carbon atoms, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, etc.

[0066] In this disclosure, the term "substituted" means that one or more hydrogen radicals in a given structure are replaced with the radical of a specified substituent.

[0067] In the present disclosure, a double-stranded oligonucleotide is composed of two chains, wherein the chain that binds to the target sequence is called the antisense chain or guide chain, and the other chain is called the sense chain or passenger chain. The term "antisense chain" refers to a chain of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to the target sequence. The term "sense chain" refers to a chain of a double-stranded oligonucleotide that includes a region that is substantially complementary to the region of the antisense chain as defined herein. The term "complementary region" refers to a region on the antisense chain that is completely or substantially complementary to the target sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. Herein, double-stranded oligonucleotides and siRNA can be used interchangeably.

[0068] In the present disclosure, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases on the other strand in a complementary manner.

[0069] In the present disclosure, unless otherwise specified, "substantially reverse complementary" or "substantially complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0070] In the present disclosure, the term "C4B" includes: human C4B, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:189181756; mouse C4B, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:218156288 and GI:218156290; rat C4B, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:218156284; and chimpanzee C4B, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:57114201. The term "C4B" also includes cynomolgus macaque C4B, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:544428919. Additional examples of C4B mRNA sequences are readily available, for example, using GenBank, UniProt, Online Mendelian Inheritance in Man (OMIM), and the Macaca mulatta genome sequencing project website.

[0071] Exemplary C4B nucleotide sequences of the present disclosure are listed in Tables 1-2. The term "C4B" also refers to naturally occurring DNA sequence variations of the C4B gene. It also refers to single nucleotide polymorphisms within the C4B gene. Many sequence variations within the C4B gene have been identified and can be discovered, as is known in the art.

[0072] In the present disclosure, the term "target gene" refers to a gene that transcribes and expresses C4B mRNA.

[0073] In the present disclosure, the terms "RNAi", "iRNA", "RNAi agent", "RNAi reagent", "RNA interference agent", "RNA inhibitor" are used interchangeably herein to refer to molecules or agents that contain RNA and can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. It is well known in the art that RNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). In one embodiment, the RNAi agents of the present disclosure include single-stranded or double-stranded RNA that interacts with a target RNA sequence (e.g., a C4B, C3, or C9 target mRNA sequence) to guide the cleavage of the target RNA. Therefore, in one aspect, the present disclosure relates to the term "siRNA" which can also be used to refer to the RNAi described above.

[0074] In some embodiments, the "RNAi" used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and the "RNAi agent" includes the double-stranded RNA, and the "RNAi agent" can refer to a "double-stranded RNAi agent", a "double-stranded RNA (dsRNA) molecule", a "dsRNA agent", a "siRNA agent", or a "dsRNA agent".

[0075] In the present disclosure, "dsRNA" or "siRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA (e.g., C4B, C3 or C9 gene).

[0076] In the present disclosure, as used in this specification, "RNAi agents" or "RNAi agents" may include ribonucleotides with chemical modifications and / or ligands; RNAi agents may include substantial modifications at multiple nucleotides. The term "modified nucleotides" refers to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms of internucleotide linkages, sugars, or nucleobases. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. As used in siRNA molecules, any such modification may be encompassed by "RNAi agent."

[0077] In the present disclosure, the term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. The one or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0078] In the present disclosure, the term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or binding.

[0079] In the present disclosure, the term "administer" generally refers to introducing the disclosed pharmaceutical formulation into the body of a subject by any introduction or delivery route. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug can be used. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous or oral administration. The daily dose may be divided into one, two or more doses in a suitable form for administration at one, two or more times during a certain time period.

[0080] In the present disclosure, the term "contact" generally refers to the contacting of two or more different types of substances in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to direct contact of a RNAi agent or composition of the present disclosure with a cell or tissue. In other embodiments, the term refers to indirect contact of a RNAi agent or composition of the present disclosure with a cell or tissue.

[0081] In the present disclosure, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects, and subjects include animal disease models.

[0082] In this disclosure, the term "pharmaceutical composition" or "composition" may refer to a composition for use in the treatment of a disease, as well as for use in in vitro cell culture experiments. When used in the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0083] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" in the present disclosure means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0084] In the present disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0085] In this disclosure, the terms "treat," "treat," or "ameliorate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0086] In this disclosure, the terms "prevention" and "prevent" are used interchangeably to refer to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0087] As used herein, the term "modulate gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated so that the expression, level, or activity is greater or less than that observed in the absence of the modulator. For example, the term "modulate" may mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[0088] In the present disclosure, the term "inhibiting complement C4B gene expression" is intended to refer to inhibiting the expression of any C4B gene, as well as variants or mutants of the C4B gene. Thus, the C4B gene can be a wild-type C4B gene, a mutant C4B gene, or a transgenic C4B gene in the context of a genetically manipulated cell, cell group, or organism.

[0089] "Inhibiting C4B gene expression" includes any level of inhibition of the C4B gene, such as at least partial suppression of C4B gene expression. C4B gene expression can be assessed based on the level or change in level of any variable associated with C4B gene expression, such as C4B mRNA level, C4B protein level, or lipid level. This level can be assessed in an individual cell or in a group of cells (including, for example, a sample derived from a subject).

[0090] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0091] Double-stranded oligonucleotide (dsRNA)

[0092] In a first aspect, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for inhibiting complement C4B gene expression. The double-stranded oligonucleotide is capable of inhibiting complement C4B gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide is capable of inhibiting C4B gene expression in cells in vitro and in vivo.

[0093] The double-stranded oligonucleotide includes a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides.

[0094] In some optional embodiments of the present disclosure, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO:1-SEQ ID NO:180 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO:181-SEQ ID NO:360 in Table 1.

[0095] The sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides, and the double-stranded region formed by the sense strand and the antisense strand includes at least 15 nucleotide pairs.

[0096] In some optional embodiments of the present disclosure, the sense strand comprises a nucleotide sequence that differs from any one of the sequences shown in SEQ ID NO:1-SEQ ID NO:180 in Table 1 by no more than 1 or 2 nucleotides, and / or the antisense strand comprises a nucleotide sequence that differs from any one of the sequences shown in SEQ ID NO:181-SEQ ID NO:360 in Table 1 by no more than 1 or 2 nucleotides.

[0097] In some optional embodiments of the present disclosure, the positive strand comprises a nucleotide sequence that differs from any one of the sequences shown in SEQ ID NO:1-SEQ ID NO:180 in Table 1 by no more than 1 nucleotide, and / or the antisense strand comprises a nucleotide sequence that differs from any one of the sequences shown in SEQ ID NO:181-SEQ ID NO:360 in Table 1 by no more than 1 nucleotide.

[0098] In a specific embodiment of the present disclosure, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1.

[0099] In some optional embodiments of the present disclosure, the sense strand or the antisense strand comprises any sense strand or antisense strand selected from the duplexes shown in the following numbers in Table 1: RN012001, RN012003, RN012023, RN012031, RN012051, RN012055, RN012059, RN012062, RN012075, RN012092, RN012093, RN012094, RN012103, RN012106, RN012107, RN012110, RN012111, RN012112, RN012113, RN01 2114, RN012128, RN012135, RN012136, RN012148, RN012155, RN012160, RN012161, RN012163.

[0100] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide comprises one or more of the duplex groups shown in the following numbers in Table 1: RN012001, RN012003, RN012023, RN012031, RN012051, RN012055, RN012059, RN012062, RN012075, RN012092, RN012093, RN 012094, RN012103, RN012106, RN012107, RN012110, RN012111, RN012112, RN012113, RN012 114. RN012128, RN012135, RN012136, RN012148, RN012155, RN012160, RN012161, RN012163.

[0101] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide comprises one or more selected from the duplex group shown in the following numbers in Table 1: RN012001, RN012023, RN012059, RN012106, RN012148, RN012160.

[0102] Each nucleotide of the double-stranded oligonucleotide is an unmodified or modified nucleotide.

[0103] In some optional embodiments of the present disclosure, at least one nucleotide in the double-stranded oligonucleotide is modified.

[0104] In some optional embodiments of the present disclosure, all nucleotides in the sense strand and all nucleotides in the antisense strand of the double-stranded oligonucleotide are modified nucleotides.

[0105] The modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more nucleotides substituted with OR; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0106] In some optional embodiments of the present disclosure, the modified nucleotides are selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR, R is as defined above.

[0107] In a specific embodiment of the present disclosure, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 3, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 3.

[0108] In some optional embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide comprises a sense strand nucleotide sequence selected from any one of the following modified sequences shown in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, and RZ012234; and / or, the antisense strand comprises an antisense strand nucleotide sequence selected from any one of the following modified sequences shown in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, and RZ012234.

[0109] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide comprises one or more selected from the group of modified duplexes shown in the following numbers in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, and RZ012234.

[0110] In some embodiments, the length of the antisense strand and the sense strand is 17-25 nucleotides.

[0111] In some optional embodiments of the present disclosure, at least one of the antisense strand and the sense strand comprises a 3'-end overhang of 1-2 nucleotides.

[0112] Double-stranded RNAi agents

[0113] In a second aspect of the present disclosure, the present disclosure provides a double-stranded RNAi agent for inhibiting complement C4B gene expression, characterized in that the double-stranded RNAi agent comprises a sense strand and an antisense strand, the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, and the double-stranded region is represented by the following formula (I):

[0114] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0115] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I),

[0116] Among them, SS represents the sense strand and AS represents the antisense strand;

[0117] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0118] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy-modified nucleotide;

[0119] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0120] Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10;

[0121] Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0122] wherein the nucleotide N is selected from 2'-O-methyl modified nucleotides and 2'-fluoro-modified nucleotides, and the nucleotide X is 2'-O-methyl modified nucleotides or 2'-O-methoxyethyl modified nucleotides;

[0123] In some optional embodiments of the present disclosure, the duplex comprises at least one 2'-O-methoxyethyl modification or 2'-O(CH2) n OR-modified nucleotides;

[0124] In some optional embodiments of the present disclosure, the double-stranded RNAi agent comprises the double-stranded oligonucleotide described above.

[0125] In some embodiments, the nucleotides of the sense strand and / or antisense strand of the double-stranded RNAi agent are all modified nucleotides, and: wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; and optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0126] In some embodiments, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

[0127] In some optional embodiments of the present disclosure, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 of the nucleotide sequence in the positive strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides.

[0128] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0129] In some optional embodiments of the present disclosure, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain are 2'-fluorinated modified nucleotides, and any one nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides.

[0130] In some optional embodiments of the present disclosure, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain are 2'-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

[0131] In some optional embodiments of the present disclosure, the nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4):

[0132] (1) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0133] (2) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides;

[0134] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides;

[0135] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

[0136] In a specific embodiment of the present disclosure, the sense strand is any modified sense strand nucleotide sequence shown in Table 2-3, and / or the antisense strand is any modified antisense strand nucleotide sequence shown in Table 2-3.

[0137] In some optional embodiments of the present disclosure, the sense chain is any modified sense chain nucleotide sequence shown in the following numbers in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, RZ012234; and / or, the antisense chain is any modified antisense chain nucleotide sequence shown in the following numbers in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, RZ012234.

[0138] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide is selected from one or more of the modified duplex groups shown in the following numbers in Table 3: RZ012209, RZ012211, RZ012215, RZ012222, RZ012232, and RZ012234.

[0139] In some embodiments, the double-stranded RNAi agent further comprises a ligand; in some optional embodiments of the present disclosure, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent.

[0140] In some embodiments, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives connected by a linker selected from a monovalent, divalent or trivalent branched linker.

[0141] In some embodiments, the ligand structure is as follows:

[0142] * represents the attachment site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is independently selected from hydroxyl or sulfhydryl; p is independently selected from 1, 2 or 3; q is independently selected from 1, 2 or 3; each R3 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is C1-C 20 The alkylene chain, or one or more C atoms in the chain, is substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is independently NH, O or S.

[0143] In other embodiments, the specific structure of the ligand is as follows:

[0144] In some embodiments, the 3' end of the sense strand of the double-stranded RNAi agent is covalently coupled to a ligand.

[0145] In some embodiments, the structure of the double-stranded RNAi agent of the present disclosure comprises a conjugate as shown in the following formula:

[0146] Wherein, Nu represents a double-stranded oligonucleotide molecule; in a specific embodiment, the Nu represents any modified sense chain nucleotide sequence shown in Table 2-3, and / or any modified antisense chain nucleotide sequence shown in Table 2-3.

[0147] In some alternative embodiments of the present disclosure, the ligand is linked to the 3' end of the sense strand.

[0148] In some optional embodiments, the double-stranded RNAi agent can be represented by the formula Nu-Za, wherein Nu represents a double-stranded oligonucleotide molecule and Za is a ligand with the following structure:

[0149] In some embodiments, the sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length.

[0150] In some embodiments, the sense strand of the double-stranded region is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long. Preferably, the double-stranded region is 19 to 23 nucleotide pairs long; or, the double-stranded region is 19 to 21 nucleotide pairs long.

[0151] In some embodiments, the sense strand and / or antisense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages.

[0152] In some embodiments, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends.

[0153] In some embodiments, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

[0154] In some embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein each strand is 17-25 nucleotides, and the antisense strand comprises a double-stranded region region that is complementary to a portion of an mRNA encoding complement C4B (SEQ ID NO: 361), wherein the double-stranded region is represented by the following Formula II:

[0155] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0156] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(II)

[0157] Wherein, SS represents the sense strand, AS represents the antisense strand, and the SS strand can be conjugated to a ligand.

[0158] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy modified nucleotide; each of the Xs independently represents a 2'-O-methoxyethyl modified nucleotide;

[0159] wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0, and 0≤q'+r'≤2;

[0160] Furthermore, (N)a contains at least one fluorinated-modified nucleotide, and the 16th position of the antisense strand, starting from the 5'-end, is a fluorinated-modified nucleotide; (N)b contains at least one fluorinated-modified nucleotide, and the 14th position of the antisense strand, starting from the 5'-end, is a fluorinated-modified nucleotide; (N)c contains at least two fluorinated-modified nucleotides, and positions 2 and 6 of the antisense strand, starting from the 5'-end, are both fluorinated-modified nucleotides; the first four nucleotides of (N)b', starting from the 5'-end, contain at least two fluorinated-modified nucleotides; and there is at least one X on the SS strand.

[0161] In some embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand of Formula II are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

[0162] On the other hand, the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of complement C4B, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1. Wherein, each strand is no more than 25 nucleotides in length, and both the sense strand and the antisense strand are modified nucleotides. The modified nucleotides comprise modified nucleotides selected from 2'-O-methyl modification and 2'-fluoro modification, and the sense strand comprises 1-3 phosphorothioate internucleotide bonds; wherein the modified nucleotides in the antisense strand comprise modified nucleotides selected from 2'-O-methyl modification, 2'-O-methoxyethyl modification and 2'-fluoro modification, wherein the antisense strand comprises 2-5 phosphorothioate internucleotide bonds; and wherein the sense strand is conjugated to a GalNAc ligand at the 3'-terminus.

[0163] Uses in disease treatment

[0164] In a third aspect of the present disclosure, the present disclosure provides use of any of the following in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the complement C4B gene:

[0165] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0166] (2) The double-stranded RNAi agent described in the second aspect of the present disclosure.

[0167] Pharmaceutical composition

[0168] In a fourth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any one of the following, and optionally a pharmaceutically acceptable carrier or excipient:

[0169] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0170] (2) The double-stranded RNAi agent described in the second aspect of the present disclosure.

[0171] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide and / or double-stranded RNAi agent can be administered in an unbuffered solution or a buffer, wherein the unbuffered solution is saline or water, and the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; alternatively, wherein the buffer is phosphate-buffered saline (PBS).

[0172] Reagent test kit

[0173] In a fifth aspect of the present disclosure, the present disclosure provides a kit comprising any one of the following:

[0174] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0175] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0176] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0177] Method for inhibiting complement C4B expression in cells in vitro

[0178] In a sixth aspect of the present disclosure, the present disclosure provides a method for inhibiting complement C4B expression in cells in vitro, the method comprising:

[0179] (a) contacting the cell with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition thereof; and

[0180] (b) maintaining the cells produced in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript of the complement C4B gene, thereby inhibiting expression of the complement C4B gene in the cells.

[0181] wherein the complement C4B expression is inhibited by at least 70%.

[0182] Disease treatment methods

[0183] In a seventh aspect of the present disclosure, the present disclosure provides a method for alleviating, treating and / or preventing a disease or condition mediated by complement C4B, characterized by administering any of the following to a subject:

[0184] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0185] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0186] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0187] Among them, the complement C4B-mediated diseases or conditions include diseases related to the mRNA level of complement C4B gene expression, including IgA nephropathy, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), glomerulopathy, systemic lupus erythematosus, etc.

[0188] Methods for inhibiting complement C4B gene expression

[0189] In an eighth aspect of the present disclosure, the present disclosure provides a method for inhibiting complement C4B gene expression, characterized in that the method comprises administering any of the following to a subject:

[0190] (1) the double-stranded oligonucleotide described in the first aspect of the present disclosure; and / or

[0191] (2) the double-stranded RNAi agent described in the second aspect of the present disclosure; and / or

[0192] (3) The pharmaceutical composition described in the fourth aspect of the present disclosure.

[0193] In some alternative embodiments of the present disclosure, the subject is a human or other mammal.

[0194] In some embodiments, expression of the complement C4B gene is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0195] In some embodiments, inhibiting the expression of complement C4B reduces the level of complement C4B protein in the serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0196] The double-stranded RNAi agent or pharmaceutical composition thereof provided by the present disclosure can effectively regulate the expression level of the complement C4B gene, and thus can effectively treat and / or prevent diseases or symptoms associated with dysregulated complement C4B gene expression.

[0197] In some optional embodiments of the present disclosure, the double-stranded RNAi agent or its pharmaceutical composition can be administered by any suitable means, such as in the form of an injection or infusion solution (e.g., subcutaneously, intramuscularly or intravenously), and the administration mode includes but is not limited to single administration or multiple administration; the dosage range is 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, such as 3 mg / kg, 10 mg / kg or 30 mg / kg.

[0198] In order to make the purpose, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to examples.

[0199] Exemplarily, the mRNA sequence encoding C4B of the present invention (SEQ ID NO: 361) is as follows:

[0200] In the context of the present disclosure, unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the synthesis of PCR primers used in the present disclosure was commissioned to Sangon Biotech (Shanghai) Co., Ltd.; the human liver cancer cell line HepG2 used in the present disclosure was purchased from Wuhan Prosai Life Science Co., Ltd.; the experimental animal C57BL / 6J mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.

[0201] In the context of this disclosure, unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0202] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0203] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0204] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0205] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0206] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0207] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0208] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0209] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0210] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0211] The double-stranded oligonucleotide (dsRNA) sequences described in the present disclosure are shown in Table 1.

[0212] Table 1 Unmodified double-stranded oligonucleotide sequence list

[0213] Preparation of ligands

[0214] Preparation Example 1 Preparation of CR01008 ligand

[0215] (1.1) Synthesis of compound CR01008

[0216] The synthetic route of compound CR01008 is as follows:

[0217] (1.1.1) Synthesis of Compound 2

[0218] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3% by mass KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 ° C for 30 minutes, heated to 60 ° C and refluxed at 60 ° C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry, and filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M + H] + .

[0219] (1.1.2) Synthesis of compound 3

[0220] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 mL of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 mL) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0221] (1.1.3) Synthesis of compound 5

[0222] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) prepared in step (1.1.2) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (22% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0223] (1.1.4) Synthesis of Compound 6

[0224] Compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0225] (1.1.5) Synthesis of compound CR01008

[0226] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0227] 1 H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0228] (1.2) Synthesis of compound CR01008Z

[0229] Compound CR01008Z was obtained by linking compound 6, which was used to synthesize compound CR01008, to a solid phase support CPG.

[0230] The synthetic route of compound CR01008Z is as follows:

[0231] (1.2.1) Synthesis of compound 9

[0232] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0233] (1.2.2) Synthesis of compound CR01008Z

[0234] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0235] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0236] Preparation Example 2 Preparation of (CR01008) × 3 ligand

[0237] By the phosphoramidite nucleic acid solid phase synthesis method, the solid phase support compound (CR01008Z) described in Preparation Example 1 was used as the starting cycle to connect the compounds (CR01008) that were not connected to the solid phase support one by one.

[0238] Each compound connection involves four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given below:

[0239] The compound CR01008 not bound to a solid support was separately prepared into a 0.1 M solution using acetonitrile.

[0240] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0241] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the compound attached to the solid support to the compound not attached to the solid support of 1:10, a molar ratio of the compound attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0242] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the compound attached to the solid support was 1:1:1.

[0243] The conditions for each oxidation or sulfidation reaction were identical. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; sulfidation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of sulfidation agent to the compound attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9).

[0244] Three clusters of CR01008 (denoted as (CR01008)×3 or CR01008×3) were obtained through the above method.

[0245] The structural formula of the three-cluster CR01008 is as follows:

[0246] Preparation of double-stranded RNAi agents

[0247] Preparation Example 3 Preparation of double-stranded RNAi agent with L96 as ligand

[0248] Step 1: L96-PS

[0249] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0250] Wherein, PS represents polystyrene resin solid phase carrier.

[0251] Step 2: Synthesis of the Sense Strand

[0252] Using phosphoramidite solid-phase nucleic acid synthesis, the L96-PS compound attached to a solid support is used as the initial cycle, and nucleoside monomers are attached one by one in the 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer attachment involves four steps: deprotection, coupling, capping, and oxidation or sulfurization.

[0253] Using the phosphoramidite nucleic acid solid-phase synthesis method, the solid-phase support compound is initially linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0254] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0255] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0256] The coupling reaction conditions were identical for each step. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0257] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0258] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0259] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0260] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to the solid phase support was added to 0.5 ml / μmol of 25% by mass ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0261] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0262] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC); molecular weight was detected using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value ≈ the theoretical value, it indicated that the ligand was conjugated to the 3' end of the sense strand.

[0263] Step 3: Synthesis of the antisense strand

[0264] The antisense strand was synthesized using a universal solid-phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification, and desalting conditions in the solid-phase synthesis of the antisense strand were the same as those in step 2 for the sense strand.

[0265] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value ≈ the theoretical value, the antisense strand was obtained.

[0266] Step 4: Synthesis of double-stranded RNAi agents

[0267] The sense chain synthesized in step 2 and the antisense chain synthesized in step 3 were mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining an RNAi agent having the sense chain and antisense chain shown in Table 2.

[0268] The information of the prepared double-stranded RNAi agent is shown in the following table:

[0269] Table 2 Sequence list of double-stranded RNAi agents with L96 as ligand

[0270] Preparation Example 4 Preparation of double-stranded RNAi agent with (CR01008) × 3 as ligand

[0271] Step 1: (CR01008)×3 ligand was prepared by the method shown in Preparation Example 2.

[0272] Step 2: Sense strand synthesis: Using phosphoramidite nucleic acid solid-phase synthesis, the three CR01008 compounds attached to a solid support were used as the initial cycle. Nucleoside monomers were then attached one by one in the 3'-5' direction according to the nucleotide sequence. Each ligation step involved deprotection, coupling, capping, and oxidation or sulfurization. The synthesis method and conditions were similar to those described in Example 1 for sense strand synthesis.

[0273] Step 3: Synthesis of antisense strand: The antisense strand was synthesized according to the method and conditions for synthesizing the antisense strand as described in Example 1.

[0274] Step 4: Synthesis of double-stranded RNAi agent: Prepared according to the method described in Example 1.

[0275] The information of the obtained double-stranded RNAi agent is shown in the following table:

[0276] Table 3 (CR01008) × 3 is the double-stranded RNAi agent sequence list of ligands

[0277] Unless otherwise specified, the base composition and modification meanings described in the embodiments of the present disclosure are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; the lowercase letter m indicates that the nucleotide represented by the capital letter to the left is a 2'-O-methyl-modified nucleotide; the lowercase letter f indicates that the nucleotide represented by the capital letter to the left is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide represented by the capital letter to the left is a 2'-O-methoxyethyl-modified nucleotide; the lowercase letter s indicates that the two adjacent nucleotides on the left are connected by a phosphorothioate bond.

[0278] The structural formula of 2'-O-methyl modified nucleotides is

[0279] The structural formula of 2'-fluoro modified nucleotide is

[0280] The structural formula of 2'-O-methoxyethyl modified nucleotide is

[0281] Here, Base represents the nucleobase A, U, G, C, or T.

[0282] Biological detection experiments

[0283] Unless otherwise stated, the reagents and consumables (Table 4) and instruments and equipment (Table 5) used in this disclosure were all commercially available products from the following manufacturers.

[0284] Table 4 Main reagents and consumables

[0285] Table 5 Main instruments and equipment

[0286] Example 1 In vitro activity evaluation of double-stranded RNAi agents with L96 as ligand

[0287] This example uses the target gene inhibitory activity assessment method in the human liver cancer cell line HepG2 to evaluate the inhibitory activity of RZ012001 to RZ012180 on the target gene C4B in cells at the same C4B target site, with RZ000001 as a negative control;

[0288] Test sample preparation:

[0289] After centrifugation, dissolve each double-stranded RNAi agent in PBS according to the specifications of each tube to prepare a 20 μM stock solution. This stock solution was then serially diluted with PBS to a 1 μM working solution. Dose testing was performed at a final duplex concentration of 10 nM.

[0290] 96-well transfection and detection:

[0291] HepG2 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TM Culture medium was added, and the 96-well plate was then placed in an incubator for further incubation. 1 μL of the 1 μM working solution was dispersed in 9 μL of Opti-MEM to form the siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to the 96-well plate at a rate of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator for an additional 24 hours.

[0292] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0293] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primers for target gene and primers for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is shown in Table 6.

[0294] Table 6 Sequences of detection primers

[0295] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0296] Table 7 Inhibitory activity of target genes in HepG2 cells after administration of double-stranded RNAi agents

[0297] Example 2 Evaluation of the Activity of Double-stranded RNAi Agents with L96 as Ligand in Mouse Liver Primary Cells

[0298] In this example, C57BL / 6J mouse primary liver cells were used to evaluate the inhibitory activity of the same C4B target double-stranded RNAi agent with the 3' end of the sense strand conjugated to the L96 ligand on the target gene C4B, and RZ000002 was used as a negative control.

[0299] Isolation of primary mouse liver cells: Anesthetize 6-8 week old mice, expose the abdominal cavity to find the portal vein and inferior vena cava, and wash with preheated (37°C) HBSS-EDTA. 2+ and Mg 2+ Digestion was performed with 0.08% type IV collagenase in HBSS until the liver slowly rebounded from its elastic bulge and developed texture. The perfused liver was removed from the animal, and the cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue. The cells were then collected into a centrifuge tube. The cell suspension was centrifuged, resuspended in culture medium, and stained with trypan blue at a final concentration of 0.04% for 2 minutes to assess cell viability.

[0300] Cell plating: Dilute cells to an appropriate density with culture medium and plate at a rate of 2×10 4 The wells were inoculated in a 96-well plate, 100 μL / well.

[0301] Cell transfection: Add 1 μL of the diluted compound to a working concentration of 10 nM and gently rock the plate back and forth to mix. Incubate the cell plate at 37°C in a 5% CO2 incubator for 24 hours.

[0302] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0303] Use reverse transcription kit (Thermo Fisher Scientific company, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 reverse transcription primer, configure 20 μ L reverse transcription system and complete reverse transcription reaction according to the method of reverse transcription kit specification sheet record.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in mouse liver primary cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primer for target gene and primer for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is shown in Table 8.

[0304] Table 8 Sequences of detection primers

[0305] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer, 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0306] Table 9 Inhibitory activity of target genes in mouse liver primary cells after administration of double-stranded RNAi agents

[0307] The results in Table 9 and Figure 1 indicate that at a dose of 10 nM, multiple double-stranded RNAi agents derived from L96 vectors can significantly inhibit C4B mRNA expression in mouse primary liver cells.

[0308] Example 3 Evaluation of the activity of RNAi agents with L96 as ligand in C57BL / 6J mice

[0309] In this example, the mRNA inhibitory activity of the RNAi agents RZM12005, RZM12007, RZM12010, and RZM12035 with L96 as a ligand in the liver tissue of C57BL / 6J mice was evaluated, and RZ000001 was used as a negative control.

[0310] Animal grouping, drug administration and tissue sample collection:

[0311] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0312] The day of administration was recorded as day 0 (recorded as D0), and all mice in the groups were killed on the 7th day after administration (recorded as D7). The mice were grossly dissected and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored in RNA later and used to measure C4B mRNA expression levels. In this real-time fluorescence quantitative PCR method, GAPDH was used as an internal reference gene. Primers targeting the target gene and the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. See Table 10 for the sequences of the detection primers.

[0313] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0314] Table 10 Inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi agents

[0315] The results in Table 10 and Figure 2 show that, at a single dose of 3 mg / kg, RZM12005, RZM12007, RZM12010, and RZM12035 could significantly reduce the mRNA expression level in the liver tissue of C57BL / 6J mice, with an inhibitory effect of greater than 70%.

[0316] Example 4 In vitro activity evaluation of RNAi agents with CR01008×3 as ligand

[0317] This example uses a target gene inhibitory activity assessment method in a human liver cancer cell line HepG2 to evaluate the inhibitory activity of a double-stranded RNAi agent with CR01008×3 as a ligand against the target gene C4B in cells.

[0318] Test sample preparation:

[0319] After centrifugation of each siRNA test sample, add an appropriate amount of PBS according to the specifications of each tube to dissolve it and prepare a 20 μM stock solution. The stock solution is further diluted with PBS in a gradient manner to make 1 μM and 0.1 μM working solutions. Dosage tests are performed with final duplex concentrations of 10 nM and 1 nM.

[0320] 96-well transfection and detection:

[0321] HepG2 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TM The 96-well plate was then placed in an incubator and incubated for further incubation. 1 μL of the 1 μM or 0.1 μM working solution was dispersed in 9 μL of Opti-MEM to form the siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form the transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to the 96-well plate at a rate of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for an additional 24 hours.

[0322] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0323] Use reverse transcription kit (Thermo Fisher Scientific company, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 reverse transcription primer, configure 20 μ L reverse transcription system and complete reverse transcription reaction according to the method of reverse transcription kit specification sheet record.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression amount of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primer for target gene and primer for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is shown in Table 6.

[0324] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0325] Table 11 Inhibitory activity of target genes in HepG2 cells using double-stranded RNAi agents with CR01008×3 as ligands

[0326] The results in Table 11 and FIG3 show that the double-stranded RNAi agent with CR01008×3 as a ligand can significantly reduce the mRNA expression level in HepG2 cells.

[0327] The above specific embodiments are merely illustrative of the present invention and do not limit the present invention. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present invention, and such modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A double-stranded oligonucleotide for inhibiting complement C4B gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides in any sequence shown in SEQ ID NO:1-SEQ ID NO:180 in Table 1 or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or the antisense strand comprises at least 15 consecutive nucleotides in any sequence shown in SEQ ID NO:181-SEQ ID NO:360 in Table 1 or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; Optionally, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1; and / or, the antisense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1; in, The antisense strand is complementary or substantially complementary to the sense strand; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO:1 to SEQ ID NO:180 in Table 1 by no more than 1 or 2 nucleotides, and / or the antisense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO:181 to SEQ ID NO:360 in Table 1 by no more than 1 or 2 nucleotides; Optionally, the sense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1; Optionally, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 180 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 181 to SEQ ID NO: 360 in Table 1. Optionally, the sense strand or the antisense strand comprises any sense strand or antisense strand nucleotide sequence selected from the duplexes shown in the following numbers: RN012001, RN012003, RN012023, RN012031, RN012051, RN012055, RN012059, RN012062, RN012075, RN012092, RN012093, RN012094, RN012095, RN012096, RN012097, RN012098, RN012099, RN012010, RN012011, RN012013, RN012014, RN012015, RN012016, RN012017, RN012018, RN012019, RN012020 2094, RN012103, RN012106, RN012107, RN012110, RN012111, RN012112, RN012113, RN0121 14. RN012128, RN012135, RN012136, RN012148, RN012155, RN012160, RN012161, RN012163.

3. The double-stranded oligonucleotide according to any one of claims 1 to 2, characterized in that The double-stranded oligonucleotide comprises one or more selected from the group of duplexes shown by the following numbers: RN012001, RN012003, RN012023, RN012031, RN012051, RN012055, RN012059, RN012062, RN012075, RN012092, RN012093, RN012094, RN012103, RN012106, RN012107, RN012110, RN012111, RN012112, RN012113, RN012114, RN012128, RN012135, RN012136, RN012148, RN012155, RN012160, RN012161, and RN012163.

4. The double-stranded oligonucleotide according to any one of claims 1 to 3, characterized in that All nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides; Optionally, the length of the antisense strand and the sense strand is 17-25 nucleotides; Optionally, at least one of the antisense strand and the sense strand comprises a 3'-terminal overhang of 1-2 nucleotides. Optionally, the modified nucleotide is selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino; Optionally, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, Decorative nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; Optionally, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 3, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 3.

5. A double-stranded RNAi agent for inhibiting complement C4B gene expression, characterized in that: The double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the double-stranded region is represented by the following formula (I): SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (I), Among them, SS represents the sense strand and AS represents the antisense strand; All nucleotides of the sense strand and the antisense strand are modified nucleotides; The Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituted substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino; Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

6. The double-stranded RNAi agent according to claim 5, characterized in that a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; wherein the nucleotide N is selected from 2'-O-methyl modified nucleotides and 2'-fluoro-modified nucleotides, and the nucleotide X is a 2'-O-methoxyethyl modified nucleotide; Optionally, the duplex includes at least one 2'-O-methoxyethyl modification or 2'-O(CH2) n OR-modified nucleotides; Optionally, the double-stranded RNAi agent comprises the double-stranded oligonucleotide of claims 1-4.

7. The double-stranded RNAi agent according to any one of claims 5 to 6, characterized in that The nucleotides of the double-stranded RNAi sense strand and / or antisense strand are all modified nucleotides; Wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Furthermore, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

8. The double-stranded RNAi agent according to claim 7, characterized in that In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; Furthermore, the nucleotides at positions 2, 6, 14, and 16 in the antisense strand are 2'-fluoro-modified nucleotides, and: any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl-modified nucleotide, and the remaining positions are 2'-O-methyl-modified nucleotides.

9. The double-stranded RNAi agent according to any one of claims 5 to 6, characterized in that The nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4): (1) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated modified nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated modified nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides.

10. The double-stranded RNAi agent according to any one of claims 5 to 9, characterized in that The double-stranded RNAi agent further comprises a ligand; Optionally, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent; Optionally, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a linker.

11. The double-stranded RNAi agent according to claim 10, characterized in that The ligands are as follows: * represents the connection site for connecting the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is each independently selected from hydroxyl or thiol; p is each independently selected from 1, 2 or 3; q is each independently selected from 1, 2 or 3; each R3 is each independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is a C1-C20 alkylene chain, or one or more C atoms in the chain are substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is each independently NH, O or S.

12. The double-stranded RNAi agent according to claim 10 or 11, characterized in that The ligand structure is as follows:

13. The double-stranded RNAi agent according to any one of claims 5 to 12, characterized in that The double-stranded RNAi agent structure is shown below: Wherein, Nu represents a double-stranded oligonucleotide molecule; Optionally, the Nu represents any modified sense strand nucleotide sequence shown in Table 2-3, and / or, any modified antisense strand nucleotide sequence shown in Table 2-3; Optionally, the ligand is attached to the 3' end of the sense strand.

14. The double-stranded RNAi agent according to claim 9, characterized in that The 3' end of the sense strand in the double-stranded RNAi agent is covalently coupled to a ligand with the following structure:

15. The double-stranded RNAi agent according to claim 14, characterized in that The double-stranded RNAi agent can be represented as Nu-Za, wherein Nu represents a double-stranded oligonucleotide molecule and Za is a ligand of the following structure:

16. The double-stranded RNAi agent according to any one of claims 5 to 15, characterized in that: The sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length; Optionally, the sense strand is 17-21 nucleotides long and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand and / or antisense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends; Optionally, the double-stranded region is 19 to 23 nucleotide pairs in length; or, the double-stranded region is 19 to 21 nucleotide pairs in length; Optionally, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

17. The double-stranded RNAi agent of claim 5, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein each strand is 14 to 25 nucleotides in length, and the antisense strand comprises a region complementary to a portion of an mRNA encoding complement C4B, wherein the double-stranded region is represented by the following Formula II: SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (II) in, SS represents the sense strand, and AS represents the antisense strand; each of the Ns independently represents a nucleotide modified as follows: a 2'-O-methyl modified nucleotide, a 2'-fluorine modified nucleotide, or a 2'-deoxy-modified nucleotide; each of the Xs independently represents a 2'-O-methoxyethyl modified nucleotide; wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0 at the same time, 0≤q'+r'≤2; Furthermore: (N)a contains at least one fluorinated modified nucleotide, and the 16th position of the antisense strand is a fluorinated modified nucleotide starting from the 5'-end; (N)b contains at least one fluorinated modified nucleotide, and the 14th position of the antisense strand is a fluorinated modified nucleotide starting from the 5'-end; (N)c contains at least two fluorinated modified nucleotides, and the 2nd and 6th positions of the antisense strand are both fluorinated modified nucleotides starting from the 5'-end; (N)b' contains at least two fluorinated modified nucleotides in the first four nucleotides starting from the 5'-end; and there is at least one X on the SS strand; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides. Wherein, the sense strand is conjugated with a ligand.

18. The double-stranded RNAi agent of claim 17, wherein the ligand structure is as follows:

19. Use of the double-stranded oligonucleotide according to any one of claims 1 to 4 and the double-stranded RNAi agent according to any one of claims 5 to 18 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the complement C4B gene.

20. A pharmaceutical composition, characterized in that The invention comprises the double-stranded oligonucleotide according to any one of claims 1 to 4 and the double-stranded RNAi agent according to any one of claims 5 to 18, and further comprises pharmaceutically optional auxiliary materials.

21. A kit, characterized in that The kit comprises the double-stranded oligonucleotide according to any one of claims 1 to 4 and the double-stranded RNAi agent according to any one of claims 5 to 18 or a pharmaceutical composition thereof.

22. A method for inhibiting complement C4B gene expression, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 4, or a double-stranded RNAi agent according to any one of claims 5 to 18, or a pharmaceutical composition according to claim 20; Optionally, expression of complement C4B is inhibited by at least 50%, 60%, 70%, 80%, 90% or 95%; Optionally, wherein inhibiting the expression of complement C4B reduces the level of complement C4b protein in the serum of the subject by at least 50%, 60%, 70%, 80%, 90% or 95%.

23. A method for alleviating, treating and / or preventing a disease or condition mediated by complement C4B, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 4, or a double-stranded RNAi agent according to any one of claims 5 to 18, or a pharmaceutical composition according to claim 20; Optionally, the complement C4B-mediated disease or disorder includes a disease associated with the level of mRNA expressed by the complement C4B gene; Optionally, the disease comprises IgA nephropathy, atypical hemolytic uremic syndrome (aHUS), myasthenia gravis (MG), glomerulopathy, systemic lupus erythematosus.

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